Neovascularization has been described as an early and sensitive marker for pre-clinical cardiovascular disease and, in particular, atherosclerosis. There is an association between intra-plaque neovascularization (sprouting from the vasa vasorum), intra-plaque hemorrhage (IPH), the size of the necrotic core and plaque vulnerability. Hence, there is a great clinical need for a non-invasive imaging tool to enable early detection and assessment of vulnerable plaques in individuals considered to be at risk for a future cardiovascular event. Contrast-enhanced ultrasound imaging (US) using microbubbles is one such potential tool, which has been used to image intra-plaque neovascularity. We propose to expand on this concept by utilizing contrast microbubbles as both nonlinear imaging agents and as sensors for non-invasive pressure estimation in atherosclerotic plaques. Our group has demonstrated that the nonlinear subharmonic signals from microbubble-based US contrast agents (UCAs) can be used in a new subharmonic imaging (SHI) mode. Moreover, these signals provide an excellent indication of hydrostatic pressures (errors<4 mmHg). Based on such results, an innovative and quantitative technique called subharmonic-aided pressure estimation (SHAPE) was proposed and investigated in pilot studies. This project aims to develop 3D SHI and SHAPE for the non-invasive, real time in vivo evaluation of plaque neovascularity as well as intra-plaque pressures (including the pressure gradient across the plaque cap) as novel biomarkers for the early detection of vulnerable plaques. We will also investigate different sized UCAs for plaque SHI and SHAPE. Initially, the effects of hydrostatic pressure on the subharmonic performance of micro- as well as nano-bubbles will be tested in vitro to select the best UCA for SHAPE (Specific Aim 1). Next, an improved version of the 3D SHI/SHAPE algorithm will be implemented on a state-of-the-art US scanner (Logiq 9, GE Healthcare, Milwaukee, WI) that can be used for real time, plaque neovascularity imaging and dynamic pressure measurements (Specific Aim 2). These new implementations will be tested in vivo in Watanabe Heritable Hyperlipidemic (WHHL) rabbits. Finally, we will assess whether in vivo 3D SHI/SHAPE of atherosclerotic lesions in WHHL rabbits can track differences in neovascularity and intra-plaque pressures over time using invasive pressure monitoring techniques and histology as the reference standards (Specific Aim 3). In conclusion, this project aims to fundamentally shift the clinical paradigm on early detection of vulnerable plaques by developing a novel, quantitative and innovative ultrasound based method (i.e., 3D SHI/SHAPE) for the non-invasive, real time evaluation of plaque neovascularity as well as estimates of intra-plaque pressures, as a first step towards the long-term goal of translating this method into a clinical trial of subjects presenting with carotid plaque.